An Investigation of the Feasibility of Volumetric Imaging of Fluorescent Bio-Markers Using Optical-ECT

Purpose: Toptical-ECT is a technique with potential for high resolution 3D imaging of the distribution of fluoresent biomarkers (including reporter proteins like GFP) in un-sectioned tissue samples. Accurate optical-ECT data is only feasible if the biomarkers survive an optical clearing procedure. This work presents investigates this question, and the feasibility of extracting volume metrics from optical-ECT data.

Methods: 4T1 tumors were grown in window chambers on nude mice, following an approved protocol. Tumor cells constitutively expressed RFP, and endogenously expressed GFP labeling HIF-1 transcription. Microvasculature was labeled by colloidal carbon. When the tumors were ~5-7mm, they were imaged in-vivo (in the chamber) using conventional epi-fluorescent microspcopy. Tumors were then immediately removed, optically-cleared, and imaged ex-vivo by optical-CT/ECT. Comparison of the in-vivo and ex-vivo images enabled investigation of the stability of the biomarkers through optical clearing. Volume measurements of regions expressing different markers (GFP and RFP) were generated though automatic thresholding.

Results: Biomarker expressing regions (GFP and RFP) were generally consistent between comparable optical-ECT projections and in-vivo microscopy. In some tumors, GFP and RFP expression was observed to be partially obscured in in-vivo images, due to absorption in overlying tissue. In optical-ECT views, these regions became visible, due to optical clearing. In one tumor, 31% of the gross tumor was deemed viable, as determined from RFP expression. 13% of the tumor was hypoxic as inferred from HIF-1 expression. Almost all the GFP hypoxic volume was within the viable RFP tumor volume.

Conclusion: Our preliminary data supports several key concepts: fluorescent biomarkers can survive the optical clearing process representative of in-vivo condition; the cleared tumor revealed new regions of signal that were partially obscured in in-vivo images; and 3D quantitative metrics can be determined from optical-CT/ECT that correspond to their 2D counterparts in standard microscopy (e.g. sub-volume of HIF-1 expression).